Anti-SESN2/Sestrin-2 antibody [EPR18907]

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-SESN2/Sestrin-2 antibody [EPR18907] (AB178518)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HCT 116 (Human colorectal carcinoma cell line) cells labeling SESN2/Sestrin-2 with ab178518 at 1/100 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining on HCT116 cells. The nuclear counter stain is DAPI (blue). Tubulin is detected with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) at 1/250 dilution (red).

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-SESN2/Sestrin-2 antibody [EPR18907] (AB178518)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed HCT 116 (Human colorectal carcinoma cell line) cells labeling SESN2/Sestrin-2 with ab178518 at 1/60 dilution (red) compared with a rabbit monoclonal IgG isotype control (ab172730; black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody; blue). Goat anti rabbit IgG (Alexa Fluor^® 488) at 1/2000 dilution was used as the secondary antibody.

• IP

Supplier Data

Immunoprecipitation - Anti-SESN2/Sestrin-2 antibody [EPR18907] (AB178518)

SESN2/Sestrin-2 was immunoprecipitated from 0.35 mg of HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate with ab178518 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab178518 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1 : HeLa whole cell lysate, 10μg (Input).

Lane 2 : ab178518 IP in HeLa whole cell lysate.

Lane 3 : Rabbit IgG,monoclonal [EPR25A] - Isotype Control (ab172730) instead of ab178518 in HeLa whole cell lysate.

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

Exposure time : 3 minutes.

SESN2/Sestrin-2 expression is low in HeLa cells and can be enriched through immunoprecipitation.

All lanes:

Immunoprecipitation - Anti-SESN2/Sestrin-2 antibody [EPR18907] (ab178518)

Predicted band size: 54 kDa

false

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-SESN2/Sestrin-2 antibody [EPR18907] (AB178518)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed NIH/3T3 (Mouse embryonic fibroblast cell line) cells labeling SESN2/Sestrin-2 with ab178518 at 1/60 dilution (red) compared with a Rabbit IgG,monoclonal[EPR25A]-Isotype control (ab172730) (black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat anti Rabbit IgG (Alexa Fluor^® 488) at 1/2000 dilution was used as the secondary antibody.

• WB

Supplier Data

Western blot - Anti-SESN2/Sestrin-2 antibody [EPR18907] (AB178518)

Blocking/Dilution buffer : 5% NFDM/TBST.

Sestrin expression is induced by H₂O₂ treatment, which is consistent with what has been described in the literature (PMID : 25337554).

Exposure time : 3 minutes

All lanes:

Western blot - Anti-SESN2/Sestrin-2 antibody [EPR18907] (ab178518) at 1/1000 dilution

Lane 1:

Untreated HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg

Lane 2:

HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate treated with 10 mM H2O2 for 1 hour at 20 µg

Lane 3:

LoVo (Human colorectal adenocarcinoma cell line) whole cell lysate at 20 µg

Lane 4:

293T (Human epithelial cell line from embryonic kidney) whole cell lysate at 20 µg

Lane 5:

NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 54 kDa

Observed band size: 54 kDa

false

Exposure time: 3min

• WB

Supplier Data

Western blot - Anti-SESN2/Sestrin-2 antibody [EPR18907] (AB178518)

Blocking/Dilution buffer : 5% NFDM/TBST.

Exposure times : Lane 1, 2, 3, 4 and 5 : 3 minutes; Lane 6 : 30 seconds.

All lanes:

Western blot - Anti-SESN2/Sestrin-2 antibody [EPR18907] (ab178518) at 1/1000 dilution

Lane 1:

HCT 116 (Human colorectal carcinoma cell line) whole cell lysate at 20 µg

Lane 2:

Rat1 (Rat fibroblast cell line) whole cell lysate at 20 µg

Lane 3:

C6 (Rat glial tumor cell line) whole cell lysate at 20 µg

Lane 4:

RAW 264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate at 20 µg

Lane 5:

Mouse spleen lysate at 20 µg

Lane 6:

PC-12 (Rat adrenal gland pheochromocytoma cell line) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 54 kDa

Observed band size: 54 kDa

false

• WB

Supplier Data

Western blot - Anti-SESN2/Sestrin-2 antibody [EPR18907] (AB178518)

Blocking/Dilution buffer : 5% NFDM/TBST.

Exposure time : 3 minutes

All lanes:

Western blot - Anti-SESN2/Sestrin-2 antibody [EPR18907] (ab178518) at 1/1000 dilution

Lane 1:

Human colon lysate at 20 µg

Lane 2:

Human fetal liver lysate at 20 µg

Lane 3:

Human testis lysate at 20 µg

Lane 4:

Human fetal kidney lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG Peroxidase Conjugate, specific to the non-reduced form of IgG at 1/10000 dilution

Predicted band size: 54 kDa

Observed band size: 54 kDa

false

Exposure time: 3min

• WB

Lab

Western blot - Anti-SESN2/Sestrin-2 antibody [EPR18907] (AB178518)

Lanes 1-4 : Merged signal (red and green). Green - ab178518 observed at 54 kDa. Red - loading control ab8245 observed at 36 kDa.

ab178518 Anti-SESN2/Sestrin-2 antibody [EPR18907] was shown to specifically react with SESN2/Sestrin-2 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265669 (knockout lysate ab257665) was used. Wild-type and SESN2/Sestrin-2 knockout samples were subjected to SDS-PAGE. ab178518 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated at room temperature for 2.5 hours at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-SESN2/Sestrin-2 antibody [EPR18907] (ab178518) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

SESN2 knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human SESN2 (Sestrin-2) knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-sesn2-sestrin-2-knockout-hela-cell-line-ab265669'>ab265669</a>)

Lane 3:

LoVo cell lysate at 20 µg

Lane 4:

HEK-293T cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/10000 dilution

Predicted band size: 38 kDa,54 kDa

Observed band size: 48 kDa,54 kDa

false

• WB

Lab

Western blot - Anti-SESN2/Sestrin-2 antibody [EPR18907] (AB178518)

False colour image of Western blot : Anti-SESN2/Sestrin-2 antibody [EPR18907] staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab178518 was shown to bind specifically to SESN2/Sestrin-2. A band was observed at 54 kDa in wild-type HEK-293 cell lysates with no signal observed at this size in SESN2 knockout cell line ab269486 (knockout cell lysate ab269650). To generate this image, wild-type and SESN2 knockout HEK-293 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4°C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged.Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

All lanes:

Western blot - Anti-SESN2/Sestrin-2 antibody [EPR18907] (ab178518) at 1/1000 dilution

Lane 1:

Wild-type HEK-293 cell lysate at 20 µg

Lane 2:

SESN2 knockout HEK-293 cell lysate at 20 µg

Lane 2:

Western blot - Human SESN2 (Sestrin-2) knockout HEK-293 cell line (<a href='/en-us/products/cell-lines/human-sesn2-sestrin-2-knockout-hek-293-cell-line-ab269486'>ab269486</a>)

Predicted band size: 54 kDa

Observed band size: 54 kDa

false

• WB

Lab

Western blot - Anti-SESN2/Sestrin-2 antibody [EPR18907] (AB178518)

Lanes 1-3 : Merged signal (red and green). Green - ab178518 observed at 54 kDa. Red - loading control ab8245 observed at 36 kDa.

ab178518 Anti-SESN2/Sestrin-2 antibody [EPR18907] was shown to specifically react with 2 in wild-type HeLa cells. Loss of signal was observed when knockout sample ab257665 was used. Wild-type and 2 knockout samples were subjected to SDS-PAGE. ab178518 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-SESN2/Sestrin-2 antibody [EPR18907] (ab178518) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

SESN2 knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human SESN2 (Sestrin-2) knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-sesn2-sestrin-2-knockout-hela-cell-line-ab265669'>ab265669</a>)

Lane 3:

HeLa cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/10000 dilution

Predicted band size: 54 kDa

Observed band size: 54 kDa

false

• WB

CiteAb

Western blot - Anti-SESN2/Sestrin-2 antibody [EPR18907] (AB178518)

SESN2/Sestrin-2 western blot using anti-SESN2/Sestrin-2 antibody [EPR18907] ab178518. Publication image and figure legend from Lees, M. J., Wilson, O. J., et al., 2020, Nutrients, PubMed 32664648.

ab178518 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab178518 please see the product overview.

Representative blots for each total and phosphoprotein. Blots are in the following order : baseline, EAA, RE, and COM. The fifth lane is occupied by the protein ladder. To the right of the experimental blots, the loading controls (calibration curves) are shown (i.e., 7.5, 10, 12.5, and 15 µL of protein).

false